熊果酸对肝纤维化大鼠肝组织TGF-β1和α-SMA表达的影响

Effects of ursolic acid on the expression of transforming growth factor-β1 and alpha-smooth muscle actin in fi brotic liver in rats

目的:观察熊果酸(UA)对肝纤维化大鼠肝组织TGF-β1基因与蛋白及α平滑肌肌动蛋白(α-SMA)表达的影响,并探讨其抗肝纤维化作用机制.方法:SD大鼠96只随机分为正常对照组(N组)、模型组(M组)、UA低剂量组(U1组)、UA中剂量组(U2组)、UA高剂量组(U3组)及秋水仙碱组(C组),每组16只.除N组外,均用二甲基亚硝胺(Dimethylnitrosamine,DMN)诱导肝纤维化4wk,分别给予安慰剂、不同剂量的UA、秋水仙碱腹腔注射,治疗4wk处死大鼠,取肝组织行病理HE染色及VG染色判断炎症和肝纤维化程度;分别采用免疫组织化学和Western blot检测TGF-β1蛋白和α-SMA蛋白的表达;采用RT-PCR检测TGF-β1mRNA的表达.结果:U2和U3组肝细胞坏死和纤维组织增生明显减轻;M组TGF-β1蛋白、TGF-β1mRNA及α-SMA蛋白较N组的表达明显增加(8.76±1.47vs1.48±0.24;0.60±0.11vs0.05±0.02;0.51±0.10vs0.09±0.02,均P<0.01).U1组和C组TGF-β1蛋白的表达较M组降低(P<0.05),U2和U3组TGF-β1蛋白的表达较M组明显降低(5.32±1.63,3.98±0.67vs8.76±1.47,均P<0.01),且低于C组的表达(7.14±1.29,P<0.05或0.01).U2和U3组的TGF-β1mRNA的表达也明显低于M组(0.36±0.07,0.25±0.06vs0.60±0.11,均P<0.01)和C组(0.47±0.10,P<0.05或0.01).U1-U3组较M组α-SMA蛋白的表达明显降低(0.36±0.08,0.23±0.02,0.15±0.03vs0.51±0.10,均P<0.01);U2和U3组α-SMA蛋白的表达也显著低于C组(0.43±0.05,均P<0.01).结论:UA能明显改善肝纤维化大鼠的肝脏组织结构,减轻肝纤维化;其抗肝纤维化的机制可能与降低TGF-β1表达,抑制HSC的激活有关.

AIM： To observe the effects of ursolic acid （UA） on the expression of transforming growth factor-β1（TGF-β1） mRNA and protein and alpha-smooth muscle actin （α-SMA） protein in liver tissue of rats with dimethylnitrosamine （DMN）-induced liver fibrosis, and explore the mechanism underlying their anti-fibrotic effects. METHODS： After liver fibrosis in rats was induced by DMN for four weeks, rats were given different doses of UA, colchicine and placebo for another four weeks by intraperitoneal injection. Hepatic lobule constitution, hepatic cell necrosis and fibrous tissue hyperplasia were observed by HE and VG staining. The levels of TGF-β1 and α-SMA proteins were measured by immunohis- tochemistry and Western blot. The expression of TGF-β1 mRNA was measured by α-PCR.RESULTS： The degree of hepatic cell necrosis and fibrous tissue hyperplasia was decreased markedly in rats treated with UA. The expres- sion levels of TGF-β1 mRNA and protein and α-SMA protein in the model control group were significantly higher than those in the normal control group （8.76±1.47 vs 1.48±0.24;0.60±0.11 vs 0.05±0.02;0.51±0.10 vs 0.09±0.02, respectively; all P〈 0.01）. The expression lev- els of TGF-β1 protein in the U1 （low-dose UA） group and colchine group were lower than those in the model control group, while the expression levels of TGF-β1 protein in the U2 （medium-dose UA） and U3 （high-dose UA） groups were not only significantly lower than that in the model control group （5.32±1.63 and 3.98±0.67 vs 8.76±1.47, both P 〈0.01）, but also lower than that in the colchine group （7.14±1.29, P 〈0.05 or 0.01）. Furthermore, the expression levels of TGF-β1 mRNA in the U2 and U3 groups were lower than those in the model control group （0.36 ±0.07 and 0.25± 0.06 vs 0.60± 0.11, both P 〈 0.01） and colchine group （0.47±0.10, P 〈 0.05 or 0.01）. The expression levels of α-SMA protein in the U1, U2 and U3 groups were significantly lower than those in the model control group （0.36±0.08, 0.23 ±0.02 and 0.15±0.03 vs 0.51±0.10, all P 〈 0.01） and colchine group （0.43± 0.05, all P 〈 0.01）. CONCLUSION： UA significantly ameliorates DMN-induced liver fibrosis perhaps through ac- tivation of HSC and downregulation of TGF-β1 expression.